Introduction: In spite of considerable progress, malaria remains a public health problem in many areas, particularly in sub-Saharan Africa. One major complexity of malaria disease is caused by the development and the spread of vector and parasite resistance to insecticides and antimalarial drugs respectively. The Pfcrt76T gene mutation has been validated as a marker conferring resistance to chloroquine and other antimalarial drugs. The extension of Plasmodium falciparum resistance to commonly used antimalarial drugs (chloroquine, sulfadoxine-pyrimethamine) led to the adoption and the use of artemisinin-based combinations in Burkina Faso since 2005. Aims: The present study was initiated to assess the prevalence of the Pfcrt76T mutation in two different malaria epidemiological setting after a decade of introduction of artemisinin-based combination therapies (ACTs) in Burkina Faso. Methodology: The study population consisted of 181 uncomplicated malaria patients recruited in Banfora and Saponé health districts in 2012 and 2013. Blood samples were collected from finger prick on filter paper, dried and sent to the Molecular Biology Laboratory at Centre National de Recherche et de Formation sur le Paludisme (CNRFP) for molecular analyzes. DNA of Plasmodium falciparum was extracted with DNA extraction kit (Qiagen®) and the Pfcrt76T mutation was determined based on Polymerase Chain Reaction / Restriction Fragment Length Polymorphism technique (RFLP). Results: The results of this study showed that the frequency of the pfcrt76T mutant allele (33.7%) was statistically lower than the Pfcrt76K wild-type allele (57.4%) in the study area. Moreover, the prevalence of Pfcrt76T mutation was neither associated with the patient age nor with the parasite density while a significant difference was observed between the two epidemiological setting, Banfora and Saponé. Conclusion: The findings of this study has shown a drop in the prevalence of mutant parasites Pfcrt76T in both the study area eight years after the introduction of ACTs compared to previous studies.